Nontypable Haemophilus influenzae is a common cause of localized respiratory tract disease, especially otitis media, sinusitis, pneumonia, and exacerbations of chronic lung disease. Following each episode of acute otitis media, fluid remains in the middle ear for weeks to months and is associated with significant hearing deficit, a sequela that in turn can impair language acquisition, speech development, and school performance. The initial step in the pathogenesis of disease due to nontypable H. influenzae involves colonization of the upper respiratory epithelium. We have demonstrated that two related high-molecular-weight proteins called HMW1 and HMW2 promote attachment to human epithelium, an essential step in the process of colonization. Examination of collections of epidemiologically unrelated strains reveals that HMW1- and HMW2-like proteins are present in 75-80% of all nontypable H. influenzae strains and are the predominant adhesins in these strains. We have established that the HMW1 and HMW2 proteins are prototype members of the two-partner secretion family and require a cognate dimeric outer membrane protein called HMW1B or HMW2B for presentation on the bacterial surface. In the process of surface localization, HMW1 and HMW2 undergo a cleavage event that eliminates a large N-terminal pro-piece. In addition, we have demonstrated that HMW1 and HMW2 are glycoproteins and are modified by a glycosyltransferase called HMW1C or HMW2C, capable of transferring hexose units to asparagine residues. Based on studies of HMW1, over 30 asparagines are modified with mono- or di-hexoses, including glucose and galactose. In the present proposal we plan to elucidate the mechanism of HMW1 and HMW2 surface localization and maturation. In particular, we will define the determinants of interaction between HMW1 and HMW1B, the importance of HMW1B dimer formation for HMW1 secretion, and the mechanism of cleavage of the HMW1 pro-piece. In additional experiments, we will elucidate the molecular details of glycosylation, defining the relationship between specific asparagine sites of glycosylation and the adhesive function of HMW1 and the relationship between glycosylation and immunogenicity of HMW1. Finally, we will characterize the HMW1C-like family of glycosyltransferases. From a practical perspective, the results of these studies may be directly relevant to the development of novel antimicrobials and vaccines effective in the treatment and prevention of nontypable H. influenzae disease. More generally, they may provide fundamental insights into host-microbial relationships, bacterial protein secretion, and prokaryotic protein glycosylation. PUBLIC HEALTH RELEVANCE: Nontypable Haemophilus influenzae is a common cause of localized respiratory tract disease, especially otitis media, sinusitis, pneumonia, and exacerbations of chronic lung disease. From a practical perspective, the results of the proposed studies may be directly relevant to the development of novel antimicrobials and vaccines effective in the treatment and prevention of nontypable H. influenzae disease. More generally, these studies will provide fundamental insights into host-microbial relationships, bacterial protein secretion, and prokaryotic protein glycosylation.